Celiac disease, also known as coeliac disease or celiac sprue (coeliac sprue), affects approximately 1% of people in Europe and North America. In many of those affected, celiac disease is unrecognised, but this clinical oversight is now being rectified with greater clinical awareness. A gluten free diet is the only current treatment for celiac disease, and because regular ingestion of as little as 50 mg of gluten (equivalent to 1/100th of a standard slice of bread) damages the small intestine, chronic inflammation of the small bowel is commonplace in subjects on a gluten free diet. Persistent inflammation of the small intestine has been shown to increase the risk of cancer, osteoporosis and death. As gluten is so widely used, for example, in commercial soups, sauces, ice-creams, etc., maintaining a gluten free diet is difficult.
Celiac disease occurs in genetically susceptible individuals who possess either HLA-DQ2 encoded by HLA-DQA1*05 and HLA-DQB1*02 (accounting for about 90% of individuals), variants of HLA-DQ2, or HLA-DQ8. Such individuals mount an inappropriate HLA-DQ2- and/or DQ8-restricted CD4+ T cell-mediated immune response to peptides derived from the aqueous-insoluble proteins of wheat flour, gluten, and related proteins in rye and barley.
All gluten proteins are considered toxic in celiac disease. In 2006, the NCBI public database Genbank included 345 entries for gluten proteins from bread-making wheat (Triticum aestivum), barley (Hordein vulgare) and rye (Secale cerale).
Predictive approaches have catalogued several hundred distinct putatively “toxic” gluten peptides based upon searches for homologues of known epitopes of intestinal T cell clones, or for gluten sequences predicted or proven to bind to HLA-DQ2 in vitro, having the motif favouring deamidation by tissue transglutaminase (tTG), and/or sequences resistant to proteolysis.
Authoritative reviews report there being fifty or so “immunodominant” T cell epitopes in gluten relevant to celiac disease. However, T cells raised against hordein or barley have not yet been studied, and HLA-DQ2-restricted T cell epitopes derived from high molecular weight (HMW) glutenin are yet to be defined.
Despite the large number of gluten peptides incriminated in celiac disease, the protease-resistant α-gliadin 33mer LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:1; α2-gliadin 56-88) deamidated by tTG: LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO:2) is widely regarded as the optimal stimulatory peptide (for intestinal T cell lines raised against protease-digested gluten) in HLA-DQ2 associated celiac disease. The underlining of Q residues in SEQ NO:1, and throughout this disclosure, indicates a glutamine residue amenable to deamidation catalysed by tTG or consistent with the amino-acid motif that predicts susceptibility to deamidation by tTG, i.e., Q−−>E.
This α-gliadin 33mer (SEQ ID NO:1; α2-gliadin 56-88) was recovered from a digestate of the recombinant α2-gliadin, it incorporates multiple overlapping epitopes previously identified using intestinal T cell clones and lines, and also fresh peripheral blood T cells from HLA-DQ2+ donors affected by celiac disease after in vivo gluten challenge. These epitopes include DQ2-α-I: PFPQPELPY (SEQ ID NO:3); DQ2-α-II: PQPELPYPQ (SEQ ID NO:4); and DQ2-α-III: PYPQPELPY (SEQ ID NO:5). Indeed, in vivo gluten challenge in HLA-DQ2+ celiac disease patients induces peripheral blood CD4+ T cells that are specific for a single 11mer sequence in the α-gliadin protein sequence, p60-70 PFPQPQLPYPQ (SEQ ID NO:6), that is optimally bioactive when flanked by three further residues at both the N- and C-terminal, α-gliadin p57-73 QLQPFPQPQLPYPQPQS (SEQ ID NO:7) and deamidated by tTG or Q65 substituted for glutamate, α-gliadin p57-73 QE65 QLQPFPQPELPYPQPQS (SEQ ID NO:8) that includes DQ2-α-I (SEQ ID NO:3) and DQ2-α-II (SEQ ID NO:4). However, there are hundreds of wheat, rye and barley gluten proteins, and the DQ2-α-I, DQ2-α-II, and DQ2-α-III epito pes together typically account for no more than half the toxic T cell stimulatory properties of gluten in HLA-DQ2+ celiac disease. Additional epitopes of relevance to celiac disease are disclosed in WO 01/25793, WO 03/104273 and WO 05/105129.
Although T cells have not been raised against barley hordein or rye secalin, proteins closely related to wheat gluten, the toxicity of barley and rye is ascribed to T cells specific for epitopes in wheat gluten, especially DQ2-α-I (SEQ ID NO:3) or DQ2-α-II (SEQ ID NO:4), that are cross-reactive with related hordein and secalin sequences deamidated by tTG, in particular PFPQPQQPF (SEQ ID NO:9) deamidated to Hα9/Sα9 PFPQPEQPF (SEQ ID NO:10; DQ2-ω-I) or PQPQQPFPQ (SEQ ID NO:11) deamidated to Hα2/Sα2 PQPEQPFPQ (SEQ ID NO:12), respectively.
Amongst authorities in the field, there is disagreement regarding the dominance, hierarchy, and redundancy of particular peptides in inducing T-cell stimulation in celiac disease.
Understanding the consistency and relative contribution of particular peptides to the T cell stimulatory capacity of gluten has application. Provided they consistently account for a substantial proportion of the T cell response to gluten, dominant T cell stimulatory peptides might alone or collectively enable the development of antigen-specific therapeutics and diagnostics.
In principle, antigen-specific therapy is an attractive strategy to treat autoimmune and allergic diseases. Whole protein-based approaches to desensitisation are effective for human allergic conditions and also treatment and prevention of autoimmunity and allograft rejection in experimental animal models. However, wider application of protein-based antigen-specific therapy has been limited by the small but recognised risk of anaphylaxis and because relevant antigens may not be suitable as pharmaceuticals or are simply not understood in sufficient detail to permit pharmaceutical development.
The risk of anaphylaxis can be minimised and problems of formulation overcome using short linear, aqueous soluble peptides, encompassing sequences from the disease-relevant antigen recognised by pathogenic CD4+ T cells. Peptide-based therapeutic vaccines are effective in inbred mouse models of autoimmunity and allograft rejection in which relevant immunodominant epitopes and their cognate CD4+ T cells are defined. However, even for strongly HLA-associated human immune diseases, identification of pathogenic CD4+ T cell epitopes with sufficient confidence to support rational drug design and pharmaceutical development has been very limited.
In many cases, this uncertainty is due to the fact that reported T cell responses in patients are at the limits of detection, usually depend upon in vitro expansion which may be primary or recall T cell responses, and can often also be found in healthy HLA matched individuals. These technical challenges have resulted in the compromise that peptide selection for therapeutic vaccines tends to be based upon in vitro binding affinity for disease-relevant HLA molecules, rather than their unequivocal definition as epitopes for immunodominant pathogenic T cells. A further consequence is that peptide-based compounds designed in this manner tend to encompass an expended cocktail of peptides. It might be expected that the larger the cocktail, the greater the likelihood of difficulties in formulation, stability and adverse effects, but also the more likely that T cells specific for peptides in the cocktail consistently make a substantial contribution to the pathogenic T cell response in patients.
Given the large number of toxic gluten peptides, the inventors have sought to identify an optimal non-redundant set of immunodominant peptides from which a minimal mixture could be selected for use in a peptide-based immunotherapy capable of modulating the immune response of an individual to gluten. The inventors have sought to identify immunodominant peptides useful in the treatment of celiac disease by specifically modifying the pathogenic T cell response to gluten and to therefore provide a vaccine effective against celiac disease. The same peptide mixture is also useful in diagnosis and monitoring immunomodulatory therapeutics in celiac disease.